Ribonucleic acid (RNA) is a biopolymer composed of individual monomers known as nucleotides. These nucleotides are composed of a ribofuranose ring, one of four bases, and a phosphate. The four bases that are most commonly found in RNA are adenosine (A), guanosine (G), cytosine (C), and uridine (U). RNA is most commonly used in the cell as a genetic code messenger RNA (mRNA) or as components in protein synthesis, i.e., ribosomal RNA (rRNA) and transfer RNA (tRNA). Ribonucleic acid has also been found to act as catalytic agents known as “ribozymes” and to interact with various proteins.
RNA has three different levels of structure: primary, secondary, and tertiary. The primary structure represents the specific sequence of the nucleotides found in a given RNA molecule. The secondary structure forms as the result of Watson-Crick pairing between the bases of the primary structure. Various secondary structures are formed from these base pairing interactions; such structures include: hairpin loops, internal loops, bulges, junctions, single strands, and double helices. The tertiary structure forms as the result of interactions between the various secondary structures. Tertiary structures include pseudoknots, triple helices, and loop-helix interactions.
Certain molecular interactions are important in the formation of the secondary and tertiary structures in RNA. Hydrogen bonding is involved in the formation of the Watson-Crick base pairs, which are most prevalent in the helical regions of normal A-form RNA. Stacking interactions between the bases of the RNA contribute to the stability of the RNA in both straight and helical RNA. The formation of non-canonical base pairs is also an important contributor to the formation of the tertiary structure.
Despite its many cellular functions, RNA has not, until recently, been focused upon as a potential target for drug intervention in certain diseases. Because of the numerous cellular functions of RNA, drug intervention at certain steps in cellular processes that involve RNA may prevent protein expression or other RNA-RNA interactions that are important for cellular function and/or bacterial or viral propagation.
Various small molecules have been studied for potential interaction with RNA. For example, the aminoglycosides are known to bind rRNA and to inhibit the group I intron self-splicing reaction and the hammerhead ribozyme cleavage reaction. The oxazolidinones have been shown to bind to the rRNA of the large ribosomal subunit of Gram-positive bacteria. This research is particularly important because Gram-positive bacteria such as Staphylococcus aureus, Streptococcus pneumoniae, and Enterococcus faecium are becoming serious health threats due to the antibiotic resistance developing in many strains of bacteria. Thus, due to the increasing trend of antibiotic resistance, more research is necessary to develop new, effective drugs for fighting antibiotic resistant microbial strains.
Certain gram-positive bacteria contain a unique mechanism which controls the transcription of certain genes coding for aminoacyl-tRNA synthetase, amino acid biosynthesis, and amino acid transport genes. These so called T-box genes are regulated by a common transcription antitermination system. The leader regions of the mRNA associated with these genes contain many conserved primary sequence and secondary structural elements. Specific secondary structural elements of this leader region form mutually exclusive structures known as the terminator and the antiterminator. The crucial element for controlling gene expression is the transcriptional terminator. Direct interaction between a cognate uncharged tRNA and the leader region of the mRNA triggers formation of an alternate antiterminator structure.
The specificity of the regulatory response is directed primarily by pairing of the anticodon of the tRNA with a single codon displayed at a precise position within the complex leader mRNA structure, i.e., Stem I. (see FIG. 1). A second pairing between the acceptor end of the tRNA and a conserved side-bulge region of the antiterminator structure is also required for readthrough of the mRNA. The interaction of the tRNA acceptor stem with the bulge of the antiterminator is accomplished through base pairing between the nucleotides of the acceptor stem and the nucleotides of the bulge. This second interaction presumably stabilizes the antiterminator, thereby promoting formation of the competing antiterminator structure over formation of the terminator structure. Formation of the terminator structure prevents readthrough of the mRNA (see FIG. 2).
RNA bulge loops are the disruption of an otherwise continuous helix by a single stranded region. Bulges are capable of distorting the helical backbone of RNA which can expose hydrogen bonding surfaces of the bases for interaction with other molecules such as proteins or small molecules. RNA bulges can result in sharp turns in the phosphate backbone, which often results in unfavorable electrostatic interactions as phosphate groups move closer to one another. These unfavorable electrostatic interactions create a unique spatial arrangement of charge to which positively charged molecules can bind.
The T-box system, originally discovered in a single gene in Bacillus subtilis, has been found in 19 transcription units in this organism, and has also been uncovered in a variety of Gram-positive bacteria, including Clostridium, Lactococcus, Lactobacillus, Enterococcus, Staphylococcus, Streptoccus, Deinococcus, Mycobacterium, and Corynebacterium. This list includes a number of important human pathogens. Given that aminoacyl-tRNA synthetase genes are essential for proper charging of the tRNA and cell viability, the T-box system, in particular the antiterminator bulge, provides an intriguing potential target for drug discovery. Thus, there is a need to identify compounds that interact with mRNA secondary structures and potentially disrupt or inhibit the formation of vital tertiary structures in mRNA.